Proceedings of the International Cyanide Detection Testing Workshop

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eview all the information is one reason I say probably – I fi nd no fl aws with the analysis technique. I do have concerns about the use of sulfuric acid possibly oxidizing thiocyanate to cyanide based on some work I did 20+ years ago. This is not likely to be an issue with drinking water but with biological samples, it is a concern.” There is almost nothing in the published scientifi c literature concerning the use of the ISE method for detection of cyanide associated with the digestion and distillation of fi sh tissue samples (Dzombak et al. 2006). Consequently, Dr. Kobelski noted the following concerns: 1) the possible oxidation of the cyanide by sulfuric acid in the digestion fl ask; 2) the lack of demonstration of adequate recovery in controlled experiments using spiked tissue samples; 3) that unexposed MAF might have background levels; 4) that the kinetics of cyanide might be too fast for cyanide to be detectable at points of export or import. Since there is no published data available, he proposed that research should be conducted to evaluate these questions. Need for a Field Test For Ports of Entry In USA The U.S. Fish and Wildlife Service (USFWS) agents present at the Cyanide Detection Workshop expressed the need for a reliable, rapid, easy to use fi eld test that could be used to detect cyanide or its byproducts at US ports of entry such as Los Angeles, San Francisco, Miami, and New York (where most marine ornamental fi shes are imported). There are a number of low-cost test kits that can measure CN - either using colorimetric methods or using ion selective electrodes (ISE) linked to portable ISE meters. The authors believe that because the CN - is metabolized to thiocyanate anion (by the enzyme rhodanese); it is unlikely that cyanide 54 ion would be detectable using the ASTM ISE method in importing countries like the USA, because of the long time delay before fi sh reach U.S. points of entry (by air). However, Dr. Kobelski felt that if the kinetics are slow enough for cyanide to be retained in the fi sh until the time of export, that it might also be possible to detect the presence of cyanide using the ISE method at points of import to the U.S.A. Theoretically is should be possible to measure SCN - in MAF because it would be retained for a longer time period. The advantage of measuring thiocyanate is that appreciable concentrations of SCN - may be found following exposure and it has a longer half-life than CN - (Logue and Hinkens 2008). Measuring Thiocyanate in Blood Two research grants were obtained by Dr. Roman Lanno at Ohio State University (OSU) to determine the feasibility of measuring SCN - in the blood of MAF after they had been imported into the U.S.A. (Lanno et al. 2002a, Lanno et al. 2002b). The objective of the proposed research (Lanno et al. 2002a, 2002b) was to assess whether plasma thiocyanate (SCN - ) could be used as a biomarker of cyanide exposure in marine aquarium fi shes captured using the cyanide squirt bottle technique. The objective of the study was to be met by conducting three experiments: i) Validate and refi ne an existing high pressure liquid chromatography (HPLC) technique for the determination of SCN - in the plasma of marine aquarium fi sh, ii) Determine the pharmacokinetics of SCN - in a marine aquarium fi sh species exposed to sublethal levels of waterborne cyanide, and; iii) Determine the toxicokinetics of SCN - in the plasma of a marine aquarium fi sh species exposed to a pulse dose of cyanide that simulated exposure during actual collection on a coral reef.
The intended products from the research at OSU were to include: i) a detailed manual documenting methods for monitoring SCN - in fi sh tissues that can be used to enforce regulations in importing and exporting countries, ii) Scientifi c publications describing the pharmaco-kinetics (uptake and depuration) of SCN - in MAF, outlining the bounds and limitations of the technique. A graduate student (Julie Koontz) commenced the research in 2003 under the direction of Dr. Lanno. She was able to measure SCN- concentrations in the blood of several MAF species (Pseudanthias pleurotaenia and Pseudanthias squamipinnis) using an HPLC method developed by Chen and Yang (1996). This HPLC method is capable of detecting thiocyanate in blood plasma at low levels (0.5 – 1 nmol mL-1). It requires derivatization of thiocyanate anion with 3-bromomethyl- 7-methoxy-1,4-benzoxazin-1-one, then separation by a reversed-phase C18 column. Quantifi cation is by HPLC with fl uorimetric detection. Koontz (pers. comm. 2005) experienced diffi culty in obtaining enough blood from the MAF, because of their small size. The original method, as described in Chen and Yang (1996), requires 0.5 mL of blood plasma. It may be possible to use less plasma by evaporating the entire 10 mL plasma eluate (rather than a 1.0 mL aliquot) to increase the quantity of thiocyanate ion present. The same change could also increase the sensitivity of the test for the 0.5 mL plasma volume and possibly extend the period after exposure that thiocyanate is detectable. For personal reasons, Ms. Koontz decided to drop out of the graduate research program at OSU. Hence, the pharmaco-kinetic research of SCN - dynamics in MAF was not completed. 55 Other Methods for Measuring Thiocyanate There are colorimetric tests for cyanide ion and the thiocyanate anion that could be applied (APHA-AWWA-WPCF 1992, ASTM 1997). It might be possible to measure the presence of SCN - in blood samples taken from fi sh using colorimetric methods. A spot test for these ions is published by the APHA- AWWA-WPCF (1998). Provided there are no interfering substances in the sample, the presence of CN - can be determined by colorimetric methods (e.g., chloramine T, pyridine barbituric acid, phenolphthalein reagents). The CN - can be masked using formaldehyde and the sample retested. This makes the spot test specifi c for SCN - . Whether this approach would work with blood samples is unknown. More research is necessary. It may be possible to measure SCN - levels in MAF after their tissues are dissolved in nitric acid and sulfuric acid. More acidic solutions are required to liberate SCN - from tissues than are required to liberate CN - . There is an ISE sold by Thermo-Fisher that can measure thiocyanate anion concentrations, after the tissues are digested. There are many colorimetric methods for SCN - and it is likely that HPLC or ion chromatography could be used. CDC has a liquid chromatographymass spectrometric (LC-MS/MS) method for the analysis of SCN - in both blood and urine for human clinical samples. It is unlikely that the thiocyanate ion can be distilled (like the ASTM ISE method used for detection of cyanide ion). More research is needed to determine whether a test for thiocyanate in MAF is feasible. Dr. Kobelski suggested that measuring thiocyanate in the blood of MAF was not feasible on a routine basis due to the low volume of blood in small ornamental fi sh. Whatever test is applied in the USA will
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Proceedings of the International Cy
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Proceedings of the Cyanide Detectio
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ACKNOWLEDGEMENTS The Proceedings of
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PREFACE The International Cyanide D
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EXECUTIVE SUMMARY The International
Page 13 and 14: ATCA) in homogenized fi sh tissue m
Page 15 and 16: Validation of the ISE method applie
Page 17 and 18: Importing countries should also req
Page 19 and 20: INTRODUCTION Cyanide fi shing is a
Page 21 and 22: through the implementation of a net
Page 23 and 24: Working Group 2 The Export Working
Page 25 and 26: o o o o Can the U.S. require verifi
Page 27 and 28: Polymeric membrane based ion select
Page 29 and 30: Goal 2: Provide recommendations on
Page 31 and 32: hands-on training that develops a d
Page 33 and 34: Goal 3: Identify research needs to
Page 35 and 36: Working Group 1 Participants Bob KO
Page 37 and 38: estimated at roughly 3 to 5 days. H
Page 39 and 40: Goal 2 : Identify steps, agreements
Page 41 and 42: 3) certifi cate, but tests positive
Page 43 and 44: Report from Working Group 3 The Par
Page 45 and 46: No. of Shipments 6,000 5,000 4,000
Page 47 and 48: Goal 3: Provide a recommendation wi
Page 49: Working Group 3 Participants Eric P
Page 53 and 54: Introduction Since the early 1960
Page 55 and 56: ainbow trout exposed to 40 mg SCN -
Page 57 and 58: Blennies, which were net-caught and
Page 59 and 60: electrical potential (similar to an
Page 61 and 62: in the Philippines under contract w
Page 63: endorsing the IMA’s use of the IS
Page 67 and 68: WPCF 1992, 1998). Both of these stu
Page 69 and 70: ASTM (1997) Standard test methods f
Page 71 and 72: Kuegsen, M., Kloock, J.P., Knobbe,
Page 73 and 74: Philippines. In Proceedings from th
Page 75 and 76: 3) 4) 5) 6) the metabolism of cyani
Page 77 and 78: or new matrices. 5.2.3 Modifi catio
Page 79 and 80: Level Two: The method should be val
Page 81 and 82: of cyanide exposure and to identify
Page 83 and 84: Special considerations for the anal
Page 85 and 86: tissue samples indispensable. Altho
Page 87 and 88: y reduction and subsequent separati
Page 89 and 90: can also be used for analysis of cy
Page 91 and 92: thiocyanate in saliva and serum bas
Page 93 and 94: are many discrepancies in the liter
Page 95 and 96: Table 3. Analytical Methods to dete
Page 97 and 98: Poisoning, In Textbook of military
Page 99 and 100: (70) Odoul, M., Fouillet, B., Nouri
Page 101 and 102: V. (1975) Stable reagents for the c
Page 103 and 104: (159) Felscher, D. and Wulfmeyer, M
Page 105 and 106: Purpose of this Document The purpos
Page 107 and 108: day. One cyanide tablet (2 g) is mi
Page 109 and 110: 10-90% following exposure to cyanid
Page 111 and 112: existing cyanide detection methods
Page 113 and 114: - Plasma lactate — elevated plasm
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Method Advantages Disadvantages Hig
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Paper Method Matrix Detection Limit
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Table 4 lists experts identifi ed t
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X Jerry Thomas CDC Em Steve Borron
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References Barber, C. and Pratt, V.
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Rates of Recovery. Environmental Ma
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COUNTRY REPORTS
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The first three of these destructiv
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enforcement is minimal. There is an
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1. Management/guidance to the Minis
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in Vietnam now and requires more ef
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Figure 2: Administrative structure
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References Baquero, J. (1999) Marin
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Acronyms • CDT • Cyanide Detect
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Law/Ordinance/Decree /Decision 52/2
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of samples required for analysis. U
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Field test kit. BFAR recognizes the
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Political Will Having planted the s
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gravely by fi shing communities. Th
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ecome the partner and counterpart o
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Ion-Selective Electrodes for Cyanid
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References Alban, J., Manipula, B.E
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levels with mV readings recorded as
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Trends Determined by Cyanide Testin
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Implementing agencies: • Quaranti
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APPENDIX
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1:30 Overview of and Potential Cyan
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Kristine Johnson Director Kingfi sh
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Assessment of U.S. Role in Trade: U
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